Cushing syndrome, a fatal disease, is suspected in many thousands of patients each year, but confirmed in only a fraction of these. This project seeks to identify accurately which patients have Cushing syndrome, to define the etiology of their disease and to treat it optimally. A major initiative in the past year has been to improve the approach to the differential diagnosis of Cushing syndrome. This effort included development of optimal criteria for interpretation of diagnostic tests that maintain 100% specificity for the diagnosis of Cushing's disease. We compared the performance of the 8 mg overnight dexamethasone suppression test in 41 patients who also received the traditional 6 day test and identified the best timepoints at which to measure cortisol for optimal diagnostic accuracy. We achieved 65 - 70% sensitivity using refined criteria for the traditional and overnight tests, and a 91% sensitivity if the tests were combined. Similar approach was used to optimize the traditional 4 day metyrapone stimulation test to develop criteria for 100% specificity in 185 patients with ACTH-dependent Cushing's syndrome. This test yielded a sensitivity of 72% using urine 17-hydroxysteroid and plasma 11-deoxycortisol endpoints. When combined with the traditional dexamethasone suppression test, sensitivity increased to 88%. These studies indicated that tests for the differential diagnosis of Cushing's syndrome can be combined to give improved sensitivity at 100% specificity. Further work will define an optimal cost-effective diagnostic strategy. In response to a report that venous sampling from the cavernous sinuses is superior to CRH-stimulated inferior petrosal sinus sampling, we also evaluated this test in 15 patients. In our hands, cavernous sinus sampling failed to diagnose correctly 20% of patients, while inferior petrosal sinus sampling correctly identified Cushing's disease in all patients.